Project Summary Cataract is a protein aggregation disease caused by crystallin protein defects in the lens. The congenital form of the disease results from crystallin gene mutations, whereas the age-related degenerative disease results after chemical modification of crystallin proteins. Cataracts are the leading cause of blindness in the world, with approximately 17 million cases per year. Currently, the only available treatment is surgery, which has proven successful. However, a significant fraction of the world population can not access surgery, and, in many cases, problems occur after surgery. Thus, a basic understanding of cataract formation is important to develop novel therapies that delay onset or slow progression. We will investigate the dynamics, structure and folding of cataract-associated D-crystallin mutants. Our aim is to elucidate the structural basis for cataract formation. We hypothesize that not random association of proteins, but specific folding intermediates are involved in aggregation. In addition to providing insight into the process of cataract formation, our studies will explore fundamental questions in protein biology. For example, the interactions that cause frustration of folding, questions about why and how intermediates are stabilized, and the processes that cause a polypeptide chain to misfold and/or aggregate rather than fold into the native state require direct experimental studies to gain new insights. The proposed research will address such outstanding issues through biophysical analyses of wild- type and disease-associated crystallin variants. Crystallins are ideally suited for detailed studies of protein aggregation: they are small; numerous X-ray structures are available; and the folding kinetics for several wild- type proteins to the native state have been investigated. NMR methods will be used to directly investigate folding transitions to obtain novel insights into the energetics of these processes and to elucidate structural details of the intermediates that cannot be obtained by any other methodologies. Our work will involve methods that allow detailed structural and dynamics characterization of proteins, primarily NMR spectroscopy and small angle X-ray scattering. In addition, we will correlate basic biophysical parameters with clinical observations. We plan to determine the three dimensional solution structures of cataract associated human D-crystallins and characterize their dynamic behavior. We will initially focus on two important cataract forming D-crystallin mutants, P23T and V75D. The former is associated with congenital cataracts in humans and the latter is a variant that has been identified to cause cataract in mice and, thus, will lend itself to follow-up studies in an animal model of cataract. We will also characterize the structure and dynamics of D-crystallin folding intermediates. Further, we will investigate whether and how a previously identified, partially folded D-crystallin intermediate causes aggregation. In particular, we will establish whether such partially folded intermediates are seeds for aggregation. This will prepare the basis for discovering small molecule inhibitors of aggregation, an approach that has already yielded some results in a number of neurodegenerative protein deposition diseases.